Despite almost 100 years of widespread immunization with BCG, tuberculosis (TB) remains one of the world's most devastating infectious diseases, killing over one million people every year. The principal reason that eradication of TB has proven to be difficult is that Mycobacterium tuberculosis (Mtb), the bacterium that causes TB, has devised strategies to evade the immune system. Infection occurs by inhalation and Mtb establishes a niche in the lung where it can persist for the lifetime of the host. An effective vaccine is urgently needed, but first we must gain a mechanistic understanding of how Mtb evades immune eradication in order to rationally devise a vaccination strategy that circumvents these maneuvers. Work in our lab has revealed a central role for Mtb-specific regulatory T cells (T regs) in dampening immunity during TB. We have discovered that Mtb epitope-specific, thymically-derived T regs expand in the lung draining lymph node and restrict the developing adaptive immune response. As a result, effector T cell arrival in the lung is delayed, allowing Mtb to replicate unabated for a prolonged period and establish a lung niche. Utilizing the experimental systems that we have developed to study Mtb-specific T regs, we will interrogate the pathogen-host interactions that govern their expansion and function. In Aim 1, we will pursue our recent discovery that the mycobacterial virulence lipid phthiocerol dimycocerosate (PDIM) dampens the inflammatory response to Mtb infection and promotes pathogen-specific T reg expansion. We will identify the dendritic cell (DC) subset(s) that drive Mtb-specific T reg expansion and determine whether PDIM promotes antigen presentation by these T reg-inducing DCs, or alternatively, changes their functional activation state. Key host molecular determinants that foster Mtb-specific T reg expansion will be identified and characterized. In Aim 2, we will define suppressive mechanisms utilized by Mtb-specific T regs, focusing on the functional consequences of cognate interactions between T regs and DCs during in vivo infection, and the role of CTLA-4 in modulating this activity. We will also globally survey the transcriptional landscape in Mtb-specific T regs, highly specialized cells that co-express the master regulator transcription factors Foxp3 and T-bet, and define the immunosuppressive pathways utilized. In Aim 3 we will assess T reg expansion induced by hypervirulent clinical Mtb isolates from the Beijing family lineage. We will examine whether these strains trigger an amplified and/or prolonged Mtb-specific T reg response, and if so, whether this response directs their hypervirulent phenotype. The role of phenolic glycolipids (PGL), a modified PDIM that bears an additional glycosylated phenolic moiety, in promoting these processes will also be examined. The basic experiments outlined in this proposal will set the stage for human studies and will illuminate pertinent questions that need to be addressed in the clinical setting. Insights into the pathogen-host interactions that drive the expansion and function of Mtb-specific T regs will provide new potential avenues to prevent TB via vaccination and host-targeted therapy.